EUROPEAN PHARMACOPOEIA 8.0Ginkgo dry extract,refined andquantifiedTESTSWater (2.2.13):maximum 100mL/kg,determined by distillation on 20.0g of the powdered herbal drug (710)(2.9.12).Total ash (2.4.16):maximum 6.0per cent.ASSAYEssential oil (2.8.12).Use 20.0g of the freshly,coarsely powdered herbal drug,a 1000mL round-bottomed flask,10drops of liquid paraffin R or other antifoam,500mL of water R as distillation liquid and 0.5mL of xylene R in the graduated tube.Distil at a rate of 2-3mL/min for 4h.04/2008:1827GINKGO DRY EXTRACT,REFINEDAND QUANTIFIED Ginkgonis extractum siccum raffinatum etquantificatumDEFINITIONRefined and quantified dry extract produced from Ginkgo leaf (1828).Content :–flavonoids,expressed as flavone glycosides (M r 756.7):22.0per cent to 27.0per cent (dried extract);–bilobalide :2.6per cent to 3.2per cent (dried extract);–ginkgolides A,B and C :2.8per cent to 3.4per cent (dried extract);–ginkgolic acids :maximum 5ppm (dried extract).PRODUCTIONThe extract is produced from the herbal drug by an appropriate procedure using organic solvents and their mixtures with water,physical separation steps as well as other suitableprocesses.CHARACTERS Appearance :bright yellow-brown,powder or friable mass.IDENTIFICATIONThin-layer chromatography (2.2.27).Test solution .Dissolve 20.0mg of the extract to be examinedin 10mL of a mixture of 2volumes of water R and 8volumesof methanol R .Reference solution .Dissolve 1.0mg of chlorogenic acid R and3.0mg of rutin R in 20mL of methanol R .Plate :TLC silica gel plate R (5-40μm)or [TLC silica gel plate R (2-10μm)].Mobile phase :anhydrous formic acid R ,glacial acetic acid R ,water R ,ethyl acetate R (7.5:7.5:17.5:67.5V/V/V/V ).Application :20μL [or 5μL],as bands.Development :over a path of 17cm [or 6cm].Drying :at 100-105°C.Detection :spray the plate whilst still hot with a 10g/L solution of diphenylboric acid aminoethyl ester R in methanol R ,then spray with a 50g/L solution of macrogol 400R in methanol R ;allow to dry in air for about 30min and examine in ultraviolet light at 365nm.Results :see below the sequence of zones present in thechromatograms obtained with the reference solution and the test solution.Furthermore,other,weaker fluorescent zones may be present in the chromatogram obtained with the test solution.Top of the plateA blue fluorescent zone Several faint coloured zones______________A brown fluorescent zone A green fluorescent zone An intense light blue fluorescent zone sometimes overlapped by a greenish-brown fluorescent zoneChlorogenic acid:a light blue fluorescent zoneOne or two green fluorescent zonesRutin:a yellowish-brown fluorescent zone One or two yellowish-brown fluorescent zones______________Several green and yellowish-brown fluorescent zonesReference solution Test solutionASSAYFlavonoids .Liquid chromatography (2.2.29).Test solution .Dissolve 0.200g of the extract to be examined in 20mL of methanol R .Add 15.0mL of dilute hydrochloric acid R and 5mL of water R and dilute to 50.0mL with methanol R .Transfer 10.0mL of this solution into a 10mL brown-glass vial.Close the vial with a tight rubber membrane stopper and secure with an aluminium crimped cap.Heat on a water-bath for 25min.Allow to cool to 20°C.Reference solution .Dissolve 10.0mg of quercetin dihydrate CRS in 20mL of methanol R .Add 15.0mL of dilute hydrochloric acid R and 5mL of water R and dilute to 50.0mL with methanol R .Column :–size :l =0.125m,Ø=4mm;–stationary phase :octadecylsilyl silica gel forchromatography R (5μm);–temperature :25°C.Mobile phase :–mobile phase A :0.3g/L solution of phosphoric acid Radjusted to pH 2.0;–mobile phase B :methanol R ;Time (min)Mobile phase A (per cent V/V )Mobile phase B (per cent V/V )0-160401-2060→4540→5520-2145→055→10021-250100Flow rate :1.0mL/min.Detector :spectrophotometer at 370nm.Injection :10μL.Relative retention with reference to quercetin (retentiontime =about 12.5min):kaempferol =about 1.4;isorhamnetin =about 1.5.System suitability :test solution:–resolution :minimum 1.5between the peaks due to kaempferol and isorhamnetin.Determine the sum of the areas including all the peaks from the peak due to quercetin to the peak due to isorhamnetin in the chromatogram obtained with the test solution (see Figure 1827.-1).General Notices (1)apply to all monographs and other texts1257Ginkgo dry extract,refined and quantified EUROPEAN PHARMACOPOEIA8.01.quercetin2.kaempferol3.isorhamnetinFigure1827.-1.–Chromatogram for the assay of flavonoids in refined and quantified ginkgo dry extract Calculate the percentage content offlavonoids,expressed asflavone glycosides,using the following expression:F1=sum of the areas of all the peaks from the peak dueto quercetin to the peak due to isorhamnetin in thechromatogram obtained with the test solution;F2=area of the peak due to quercetin in thechromatogram obtained with the referencesolution;m1=mass of quercetin dihydrate CRS in the referencesolution,in grams;m2=mass of the extract to be examined used to preparethe test solution,in grams;p=percentage content of anhydrous quercetin inquercetin dihydrate CRS.Terpene lactones.Liquid chromatography(2.2.29).Test solution.Place0.120g of the extract to be examinedin a25mL beaker and dissolve it in10mL of phosphatebuffer solution pH5.8R by stirring.Transfer the solutioninto a chromatography column,about0.15m long and about30mm in internal diameter,containing15g of kieselguhr forchromatography R.Wash the beaker with2quantities,eachof5mL,of phosphate buffer solution pH5.8R and transferthe washings to the chromatography column.Allow to standfor15min.Elute with100mL of ethyl acetate R.Evaporatethe eluate to dryness at a pressure not exceeding4kPa in awater-bath at50°C.The residue of solvent is eliminated by anair-current.Take up the residue in2.5mL of the mobile phase.Reference solution(a).Dissolve30.0mg of benzyl alcohol CRSin the mobile phase and dilute to100.0mL with the mobilephase.Reference solution(b).Place0.120g of the ginkgo dry extractfor peak identification CRS in a25mL beaker and dissolve it in10mL of phosphate buffer solution pH5.8R by stirring,thenproceed as described for the test solution.Column:–size:l=0.25m,Ø=4mm;–stationary phase:octylsilyl silica gel for chromatography R(5μm);–temperature:25°C.Mobile phase:tetrahydrofuran R,methanol R,water R(10:20:75V/V/V).Flow rate:1.0mL/min.Detection:refractometer maintained at35°C.Injection:100μL.Identification of peaks:use the chromatogram suppliedwith ginkgo dry extract for peak identification CRS and thechromatogram obtained with the reference solution(b)toidentify the peaks due to bilobalide and ginkgolides A,Band C.System suitability:–the chromatogram obtained with reference solution(b)is similar to the chromatogram supplied with ginkgo dryextract for peak identification CRS.Calculate the percentage content of bilobalide,using thefollowing expression:1258See the information section on general monographs(cover pages)EUROPEAN PHARMACOPOEIA 8.0GinkgoleafCalculate the percentage content of ginkgolide A,using the following expression:Calculate the percentage content of ginkgolide B,using the following expression:Calculate the percentage content of ginkgolide C,using the following expression:F 1=area of the peak due to bilobalide in thechromatogram obtained with the test solution;F 2=area of the peak due to ginkgolide A in the chromatogram obtained with the test solution;F 3=area of the peak due to ginkgolide B in the chromatogram obtained with the test solution;F 4=area of the peak due to ginkgolide C in the chromatogram obtained with the test solution;F 5=area of the peak due to benzyl alcohol in the chromatogram obtained with reference solution (a);m 1=mass of benzyl alcohol CRS in reference solution (a),in grams;m 2=mass of the extract to be examined used to prepare the test solution,in grams;p=percentage content of benzyl alcohol in benzyl alcohol CRS .Calculate the percentage content of the sum of ginkgolides A,B and C,using the following expression:G A =percentage content of ginkgolide A;G B =percentage content of ginkgolide B;G C=percentage content of ginkgolide C.Ginkgolic acids .Liquid chromatography (2.2.29).Test solution .Dissolve 0.500g of the powdered extract to be examined in 8mL of methanol R ,sonicating if necessary,and dilute to 10.0mL with the same solvent.Centrifuge if necessary.Reference solution .Dissolve 10.0mg of ginkgolic acids CRS in 8mL of methanol R ,sonicating if necessary,and dilute to 10.0mL with the same solvent.Dilute 2.0mL of this solution to 10.0mL with methanol R .Column :–size :l =0.25m,Ø=4.6mm;–stationary phase :octylsilyl silica gel for chromatography R (5μm);–temperature :35°C.Mobile phase :–mobile phase A :dilute 0.1mL of trifluoroacetic acid R to 1000mL with water R ;–mobile phase B :dilute 0.1mL of trifluoroacetic acid R to 1000mL with acetonitrile R ;Time (min)Mobile phase A (per cent V/V )Mobile phase B (per cent V/V )0-3025→1075→9030-35109035-3610→2590→7536-452575Flow rate :1.0mL/min.Detection :spectrophotometer at 210nm.Injection :50μL.Identification of components :use the chromatogram supplied with ginkgolic acids CRS and the chromatogram obtained with the test solution to identify the peaks due to ginkgolic acids C13,C15and C17.System suitability :reference solution:–resolution :minimum 2.0between the peaks due to ginkgolic acids C13and C15;–symmetry factor :0.8to 2.0for the peaks due to ginkgolic acids C13,C15and C17.Calculate the content in parts per million of ginkgolic acids expressed as ginkgolic acid C17,using the following expression:A 1=sum of the areas of the peaks due to the ginkgolic acids C13,C15and C17in thechromatogram obtained with the test solution;A 2=area of the peak due to ginkgolic acid C17in the chromatogram obtained with the reference solution;m 1=mass of the extract to be examined used to prepare the test solution,in grams;m 2=mass of ginkgolic acids CRS used to prepare the reference solution,in grams;p=percentage content of ginkgolic acid C17in ginkgolic acids CRS .01/2011:1828GINKGO LEAF Ginkgonis foliumDEFINITIONWhole or fragmented,dried leaf of Ginkgo biloba L.Content :not less than 0.5per cent of flavonoids,expressed as flavone glycosides (M r 757)(dried drug).IDENTIFICATIONA.The leaf is greyish or yellowish-green or yellowish-brown.The upper surface is slightly darker than the lower surface.The petioles are about 4-9cm long.The lamina is about 4-10cm wide,fan-shaped,usually bilobate or sometimes undivided.Both surfaces are smooth,and the venation dichotomous,the veins appearing to radiate from the base;they are equally prominent on both surfaces.The distal margin is incised,irregularly and to different degrees,and irregularly lobate or emarginate.The lateral margins are entire and taper towards the base.B.Reduce to a powder (355)(2.9.12).The powder is greyish or yellowish-green or yellowish-brown.Examine under a microscope using chloral hydrate solution R .The powder shows the following diagnostic characters (Figure 1828.-1):irregularly-shaped fragments of the lamina[A,B,D,E],with the upper epidermis,in surface view [D]and transverse section [E],consisting of elongated cells with irregularlyGeneral Notices (1)apply to all monographs and other texts1259。